THE REFLEX RESPONSES IN HUMAN MASTICATORY SYSTEM: MASSETER MUSCLE SINGLE MOTOR UNIT AND BITE FORCE REFLEXES

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Paulius Uginčius

THE REFLEX RESPONSES

IN HUMAN MASTICATORY SYSTEM:

MASSETER MUSCLE SINGLE MOTOR

UNIT AND BITE FORCE REFLEXES

Doctoral Dissertation Biomedical Sciences, Odontology (07B)

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The doctoral dissertation was prepared during 2010–2015 at the Institute of Physiology and Pharmacology of Lithuanian University of Health Sciences.

Scientific Supervisor

Prof. Dr. Edgaras Stankevičius (Lithuanian University of Health Scien-ces, Biomedical ScienScien-ces, Biology – 01B)

Dissertation will be defended at the Odontology Research Council of the Lithuanian University of Health Sciences:

Chairman

Prof. Dr. Antanas Šidlauskas (Lithuanian University of Health Sciences, Biomedical Sciences, Odontology – 07B)

Members:

Prof. Dr. Jurgina Sakalauskienė (Lithuanian University of Health Scien-ces, Biomedical ScienScien-ces, Odontology – 07B)

Prof. Dr. Vytenis Arvydas Skeberdis (Lithuanian University of Health Sciences, Biomedical Sciences, Biology – 01B)

Prof. Dr. Arvydas Stasiulis (Lithuanian Sports University, Biomedical Sciences, Biology – 01B)

Prof. Dr. Una Soboleva (Rīga Stradiņš University, Biomedical Sciences, Odontology – 07B)

Dissertation will be defended at the open session of the Odontology Research Council of Lithuanian University of Health Sciences at the Center of Modern Technologies of Farmacy and Health on the 31th of August, 2015 at 12.00 at noon at the Lecture Hall No. 204.

Address:

Eivenių g. 4, LT-50009 Kaunas, Lithuania.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Paulius Ugin

čius

ŽMOGAUS KRAMTYMO SISTEMOS

REFLEKSINIAI ATSAKAI:

KRAMTOMOJO RAUMENS MOTORINIŲ

VIENETŲ IR SUKANDIMO JĖGOS

REFLEKSAI

Daktaro disertacija Biomedicinos mokslai, odontologija (07B) Kaunas, 2015 3

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Disertacija rengta 2010–2015 metais Lietuvos sveikatos mokslų universiteto Medicinos akademijos Fiziologijos ir farmakologijos institute.

Mokslinis vadovas

Prof. dr. Edgaras Stankevičius (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, biologija – 01B)

Disertacija ginama Lietuvos sveikatos mokslų universiteto Medicinos akademijos Odontologijos mokslo krypties taryboje:

Pirmininkas

Prof. dr. Antanas Šidlauskas (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, odontologija – 07B)

Nariai:

Prof. dr. Jurgina Sakalauskienė (Lietuvos sveikatos mokslų universitetas, biomedicinos mokslai, odontologija – 07B)

Prof. dr. Vytenis Arvydas Skeberdis (Lietuvos sveikatos mokslų uni-versitetas, biomedicinos mokslai, biologija – 07B)

Prof. dr. Arvydas Stasiulis (Lietuvos sporto universitetas, biomedicinos mokslai, biologija – 01B)

Prof. dr. Una Soboleva (Rygos Stradinio universitetas, biomedicinos mokslai, odontologija – 07B)

Disertacija ginama viešame odontologijos mokslo krypties tarybos posėdyje 2015 m. rugpjūčio 31 d. 12:00 val. Lietuvos sveikatos mokslų universiteto Naujausių farmacijos bei sveikatos technologijų centro 204 auditorijoje. Disertacijos gynimo vietos adresas:

Eivenių g. 4, LT-50009 Kaunas, Lietuva

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ABBREVIATIONS

CPG – central pattern generator CUSUM – cumulative sum

E1, E2, and E4 – 1st, 2nd, and 4th excitation EMG – electromyogram

EPSP – excitatory postsynaptic potential I1 and I2 – 1st and 2nd inhibition

IPSP – inhibitory postsynaptic potential LA – local anaesthesia (-thetic)

SEMG – surface electromyogram (-graphic)

MacroRep – the representation of a single motor unit in the macro-EMG record

MS – muscle spindle

PMR – periodontal mechanoreceptor PSP – postsynaptic potential

SMU – single motor unit

PSF – peristimulus frequencygram PSTH – peristimulus time histogram Supr V N – supratrigeminal nucleus

TMD – temporomandibular dysfunction (-order) V Gang – trigeminal ganglion

V Mes N – trigeminal mesencephalic nucleus V Mot N – trigeminal motor nucleus

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AIM AND GOALS

Aim

To determine the importance of the periodontal mechanoreceptors (PMRs) on the control of human mastication by investigating the reflex responses of the human jaw muscles using single motor unit (SMU) and bite force recordings.

The principal hypothesis in this dissertation was that the previously-established neuronal pathways that connect trigeminal sensory receptors to the motoneurones that innervate masseter muscle are likely to be incorrect. It is further hypothesised that the inhibitory and the excitatory effects of the PMRs are underestimated.

Goals

1. To activate the PMRs and the masseter muscle spindles (MSs) simul-taneously via fast mechanical teeth stimulation and to determine the extent of the resultant reflex responses of the masseter muscle SMUs under static conditions.

2. To activate the PMRs via slow mechanical teeth stimulation and to determine the extent of the resultant masseter SMUs reflex responses under static conditions.

3. We aimed to determine the strength of the bite force reflex during rapid voluntary clench.

4. To determine if the change of a bite force rate during a sudden clench can modulate bite force reflex.

5. To evaluate the occlusal scanning system for the availability to determine jaw reflexes in clinical practice.

6. To establish neuronal pathways that connect trigeminal sensory receptors to the motoneurones which innervate masseter muscle.

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SCIENTIFIC NOVELTY

Investigation of the trigeminal reflexes in humans includes the short reflex pathways, the limitations of the surface electromyogram (SEMG) recording and the anatomical features of the masseter muscle, all of which form methodological challenges unique to the orofacial region (O‘Connor and Türker 2001). One of the obstacles of the human jaw reflex investi-gation is the difficulty to control the oral stimulations, especially mechanical stimulations for the activation of one group of the mechanoreceptors. Hence, this problem may lead to erroneous conclusions about the receptor‘s inhi-bitory or excitatory effects on jaw muscle motoneurones. When a stimulus is not well controlled (called a non-selective stimulus) in its parameters which are intensity, location, profile and duration, it can stimulate various receptor groups within the facial area, therefore eliciting various jaw muscle reflex responses. As a result, a stimulation aiming to evoke a jaw reflex may produce erroneous results about the predicted receptor contribution.

In Paper I, we have used computer controlled experimental set-up via feedback vibrator system with the preload for the mechanical teeth stimu-lation (Türker et al. 2004). This ensured us that an exact stimulus profile during all the experiment was applied to the teeth. Thereby, different PMR groups could be stimulated selectively. If a rapid-raise stimulus was used, fast adapting PMRs were activated together with the jaw MSs. If a slow-raise stimulus was used to stimulate a tooth, then slow adapting PMRs were stimulated. Accordingly, we could investigate the contributions of fast and slow adapting PMRs or the MSs to the human jaw reflexes and then we were able to evaluate the role of those receptors during mastication. Furthermore, we can understand more how human masticatory control is functioning during normal or pathological conditions such as temporo-mandibular dysfunctions (TMD), trigeminal neuralgias, bruxism, prostho-dontic patients with different kinds of prostheses, ect.

Another problem in the field of human masticatory control is that the classical analysis methods for the SEMG and/or the SMU recordings have serious methodological problems which lead to misinterpretation of the jaw-muscle activity during the reflex response. Unfortunately, the false conclu-sions about the reflex activities of jaw muscles appear in majority of studies within this field because of the wide use of these techniques separately or together without precautions and awareness of the methodological errors. Therefore, by using only the SEMG or the PSTH for the human jaw reflex experiments, the masticatory control of human cannot be understood in a synaptic level, which indicates a jam of the development of investigations in

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masticatory control studies. Notwithstanding, a novel PSF technique (Tür-ker and Cheng 1994; Tür(Tür-ker and Powers 2003, 2005) which is free from methodological errors allows us to determine PMR and masseter MS synap-tic connections to the masseter motoneurones in humans, which has not been done previously.

In order to study the connection of the trigeminal receptors to the masseter motoneurones, we have decided to utilize precisely controlled sti-muli to activate PMRs and MSs, and used local anesthetic (LA) blocks of the periodontium to make sure that the reflex originated from around the stimulated tooth. Furthermore, we have utilized both the classical and the frequency-based analysis methods to compare the two methods and to reduce count and synchronization type errors of the classical methods.

In the Paper II, the natural stimulation of the PMRs was achieved when teeth contacted during voluntary biting. As far as we know, this is the first study, which aimed to investigate if occlusal scanning system could record a bite force reflex. Several jaw muscles control jaw force and the net bite force output is a combination of the activation of all these muscles (Hannam and McMillan 1994). The net reflex response of the masticatory muscles might show the condition of the masticatory system and could be useful for the diagnosis of the dysfunctions of the masticatory system and the after-treatment follow-up. Therefore, routinely used clinical occlusal diagnostics system has been tested if it could be used for the daily evaluation of the jaw reflexes.

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INTRODUCTION TO THE STUDY OF HUMAN

MASTICATORY REFLEXES

Mastication is the vital function of the humans and mammals which regulates the high levels of bite forces and fine control of jaw muscles. In case of the loss of this fine control ability, improper positioning of teeth or damaging oral soft tissues with these extreme forces would lead to a danger for survival. Therefore, masticatory system is supplied with number of afferent inputs from intra- and perioral structures and efferent outputs to masticatory muscles.

Facial structures are supplied densely by the free nerve endings and receptors. Masticatory and mimic muscles also contain small number of muscle fibres in a motor unit. The rich sensory information supplying the masticatory system provides the fine control of masticatory movements as well as other functions of the face.

Masticatory system is controlled at the levels of central and peripheral nervous systems. The central pattern generator (CPG) is found in the brainstem and is the main masticatory rhythm generator (Lund 1991). Ascending and descending pathways of the cortex (such as corticobulbar tract) actively influence the voluntary part of mastication. Peripheral nervous system which includes receptors from intra- and perioral structures modulates the responses of the masticatory muscles via reflexes during the every phase of mastication. The receptors or free nerve endings of the jaw reflexes lie within: periodontium; mucosa of the lips, gingiva, palate and buccal mucosa; skin of the lips and face; masticatory muscles; temporo-mandibular joints; inner ear. Reflex regulation occurs in the brainstem level and the common output reaches masticatory muscle fibres via trigeminal motoneurones. Therefore, masticatory muscles can contract or relax, depending on their nature, the location of a stimulation and phase of mastication. The outcome of the reflex response may be a stronger biting for an efficient chewing or the drop of jaw for the protection from damaging the masticatory system.

For a long research period, jaw reflexes were studied by using SEMG technique (it was introduced in human neurophysiology studies in 1912) as a recording tool of the electrical activity of the jaw muscles during various oral stimulations. From 1920, intramuscular electromyography technique which uses the concentric needle electrodes enabled researches to record the SMU activity directly from the extrafusal fibres of a muscle. In 1962 a fine-wire electrodes could be inserted into the muscle with a hypodermic needle. The method minimized pain and displacement of the electrodes during

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muscle contractions since the needle was slowly withdrawn leaving two wires with bent ends within the muscle.

While recording with the fine-wire electrodes, with the help of sophis-ticated analysis techniques, the reflex response of a SMU represents exact pattern of the postsynaptic potentials (PSP) of a motoneurone. Furthermore, SMU responses may help for the indication of a number and the properties of the reflex neuronal pathways.

Most of the studies which used the SEMG technique described the reflex responses often wrongly and the importance of the responses was interpreted in a wrong way. For example, some ’complex’ reflex responses of a masseter muscle which were recorded by using the SEMG technique were not explained properly and with a confidence. Moreover, the surface EMG can show only the ‘surface’ of the reflex response with the contri-bution of neighboring mimic muscles and the movements of the skin cove-ring the muscle together with the surface electrodes.

If to analyze real reflex response of a motoneurone we need to deal with PSP and this can be achieved by using the SMU recordings and the PSF combined with the PSTH analysis techniques.

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1. LITERATURE REVIEW

1.1. Components of the human masticatory system

1.1.1. Jaw muscles Masseter

The masseter muscle is quadrilateral, and consists of three superimposed layers blending anteriorly.

The muscle comprises the largest superficial part, which arises via a thick, multileaved aponeurosis from the zygomatic process of the maxilla and anterior two thirds of the lower border of the zygomatic arch as far anteriorly as the zygomatic process, and that inserts from the angle of the mandible anteriorly to the ascending ramus and from the lower half of the lateral surface of the ramus of the mandible; its fibres pass downwards and backwards. The intermediate or middle part arises from the deep surface of the anterior two-thirds of the zygomatic arch and from the lower border of the posterior third and is inserted into the middle of the ramus of the mandible. The deep layer arises from the deep surface of the zygomatic arch and inserts into the upper part of the ramus of the mandible and into the coronoid process (Hannam 1994; Warwick and Williams 1971).

Superficial part of the muscle mostly is activated during tasks involving jaw elevation, elevation with protrusion, or movement on or towards the side contralateral to the muscle, while the deep fibers contribute strongly to jaw elevation, and more vigorously on jaw retrusion, on the side ipsilateral to the muscle. Graded activity, rather than sharp, all-or-none contributions from different muscle regions, is the general feature (Hannam 1994).

Temporalis

The temporalis is fan-shapped and arises from the whole of the temporal fossa (except the part formed by zygomatic bone) and from the deep surface of the temporal fascia. Its fibres converge and descend into a tendon which passes through the gap between the zygomatic arch and the side of the skull, to be attached to the medial surface, apex, anterior and posterior borders of the coronoid process, and the anterior border of the ramus of the mandible nearby down to the last molar tooth (Warwick and Williams 1971). The muscle has a superficial and deep parts and this is particularly noticeable in the anterior region, where the muscle has its greatest cross-sectional size (Hannam 1994). The anterior part of temporalis muscle elevates the jaw and

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keeps it in a rest position. The middle and the posterior parts of temporalis elevate and retract the jaw.

The lateral pterygoid

It is a short, thick muscle with two heads: an upper from the infra-temporal surface and infrainfra-temporal crest of the greater wing of the sphenoid bone, and a lower from the lateral surface of the lateral pterygoid plate, sometimes including the pyramidal process of the sphenoid. Its fibers pass downwards and laterally, to be inserted into a depression on the front of the neck of the mandible, and into articular capsule and disc of the temporo-mandibular articulation (Warwick and Williams 1971). Therefore, the muscle has a wide area of attachment that sweeps through an arc from near vertical to near horizontal. It is important to note that there are often close, interlacing connections between the deep fibers of the temporalis and the superior head of the lateral pterygoid (Hannam 1994). The muscle assists in opening the mouth by pulling forward the condyle, it also pulls the articular disc of the temporomandibular joint during the opening; when acting bilaterally in concert with the medial pterygoids it protrude the mandible (Hannam 1994; Warwick and Williams 1971).

The medial pterygoid

The medial pterygoid is a thick, quadrilateral muscle, which is attached to the medial surface of the lateral pterygoid plate and the grooved surface of the pyramidal process of the palatine bone. It also has a more superficial inferior head, which arises from the lateral surfaces of the pyramidal process of the palatine bone and tuberosity of the maxilla, and passes over the lower part of the lateral pterygoid before joining the main fiber group. Its fibres pass downwards, laterally, and backwards, and are attached via a strong tendinous lamina to the lower and back part of the medial surfaces of the ramus and angle of the mandible (Warwick and Williams 1971; Hannam 1994). The muscles elevate the mandible and together with the medial pterygoid of the ipsilateral side it produces lateral movements (Warwick and Williams 1971).

Digastric

The digastric has two bellies united by an intermediate rounded tendon. It lies below the body of the mandible, and extends, in an angled form, from the mastoid process to the chin. The posterior belly, which is longer than the

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anterior one, is attached in the mastoid notch of the temporal bone and passes downwards and forwards. The anterior belly is attached to the di-gastric fossa on the base of the mandible close to the median plane, and passes downwards and backwards. The two bellies meet in an intermediate tendon, which perforates the stylohyoid. It is held to the side of the body and the greater cornu of the hyoid by a fibrous loop, sometimes lined by a sinovial sheath. The digastrics depress the mandible and can elevate hyoid bone. They are especially activated in maximal depression (Warwick and Williams 1971).

1.1.2. Sensory receptors

Human studies show that all cutaneous receptors except the rapidly-adapting Pacinian endings are present in human facial skin and mucosa (Johansson et al. 1988). It was found that the reflex connection of oral sensory receptors to the jaw-closing muscles is inhibitory under static conditions (Godaux and Desmedt 1975).

Electrical or mechanical stimulation of orofacial afferents in bulk evokes responses in the jaw-closers. Bulk stimulation of intra- or perioral tissues excites many fibre types, and hence is not type specific. For example, it has been shown that mechanical stimulation of the teeth, no matter how carefully it is performed, stimulates the spindles in the jaw muscles as well as the PMRs (Türker and Jenkins 2000; Türker 2002). The connection between individual orofacial mechanoreceptive afferents and the moto-neurones that innervate jaw muscles do exist, because there is a strong synaptic coupling between type identified PMR afferents and masseter motoneurones (Türker et al. 2006). This finding indicates the important role of oral mechanosensitive receptors in the control of the human mastication.

Periodontal mechanoreceptors

Most human PMRs are slowly-adapting of Ruffini type (type II) (Lambrichts et al. 1992) and these receptors have very large receptive fields (Johansson and Vallbo 1983). PMRs are stretch receptors which are attached on the ligament of the periodontium of a tooth and are activated when the ligament is stretched by the bite forces, which move the tooth in its alveolar socket. PMRs supply information of the jaw forces applied to the teeth, they contribute to the development of strong and effective masticatory forces and also they synaptically modulate the output to the masseter motoneurones during mastication. Furthermore, PMRs contribute to excitatory or inhibitory effect on jaw muscles and help for efficient

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cation or for the protection of oral structures from the damage in case of an unexpected bite on a hard object. In lightly anaesthetized chewing rabbits by removing sensory peripheral feedback from the PMRs, the facilitation of the masseter muscle is greatly reduced (Lavigne et al. 1987; Morimoto et al. 1989).

The importance of the PMRs in humans is illustrated in patients with dental bridges supported by implants. Unlike subjects with healthy teeth, these patients chew with approximately the same pattern of muscle activity during the whole masticatory cycle (Grigoriadis et al. 2011; Haraldson 1983). Reduced bite forces in edentulous subjects (Haraldson et al. 1979) and bite force changes in subjects with locally anaesthetized teeth (Or-chardson and MacFarland 1980) show the importance of PMRs in the regulation of a bite force. Peri-implant site or edentulous jaw does not contain PMRs, therefore no afferent information could reach the central ner-vous system and no jaw adjustment could be performed according to bolus properties.

Adaptation and force threshold characteristics can be attributed to the spatial location of the receptor within the periodontal tissues rather than to the presence of morphologically distinct group of nerve endings (Hannam 1969). Therefore, it can be possible, that receptor adaptation characteristics are set by the binding between the receptor ending and the surrounding connective tissue rather than due to several types of receptor (Bonte 1993). Those with higher force threshold are located closer to the fulcrum (the centre of the rotation of a tooth during mechanical tooth stimulation) and whereas receptors with a lower force threshold are located nearer to the apex; therefore, it was postulated that the receptors at the apex will receive greater displacement and therefore only appear to have lower force thresholds (Linden and Millar 1988).

Inputs from the PMRs contribute to the regulation of the activity of the masticatory muscles (Trulsoon 2006). Therefore, information from PMRs is used in the fine motor control of jaw movements associated with biting, chewing or oral manipulations. Periodontal afferents respond to forces applied both axially and horizontally on a tooth (Trulsson et al. 1992) and anterior teeth are highly sensitive to most directions (reviewed in Trulsson 2006, 2007), hence excitatory reflex response can be elicited during chewing when teeth begin to bite into food. Incisors are loaded axially during the initial bite-split phase of chewing, and this force vector has labio-palatal direction, in which the forces in our study were applied. The importance of maintaining natural teeth with healthy periodontium when-ever possible is crucial for effective oral functions (Grigoriadis et al. 2011; Trulsoon 2006).

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Typically, PMRs respond to more than one tooth loading direction out of six anatomical directions (Trulsson 2006). Anterior teeth showed high sensitivity to most directions of loading, while posterior teeth sensitivity was the highest for lingual-distal directions of force applied. This distri-bution of sensitivity to loading has a functional demand, since anterior teeth manipulate food morsels and splitting them into pieces in the initial stages of food intake and bite forces are applied to anterior teeth in all directions (Trulsson 2006). Saturating PMRs compound 80% of the PMRs, while the rest PMRs are non-saturating within the anterior teeth region. Saturating PMRs show hyperbolic stimulus-response relationship and these receptors are the most sensitive to the sustained force levels below 1 N. Non-saturating PMRs, which consist 20% of all PMRs within the periodontium of anterior teeth, demonstrate linear stimulus-response relationship (Trulsson 2006). In the study of Trulsson (2006), the higher static and dynamic sensitivity of the PMRs has been found in anterior teeth and it reflects the slower and weaker forces developed by anterior teeth.

Jaw muscle spindles

MSs are muscle mechanoreceptors which are sensitive to the muscle length and change in the muscle length and they are important in the control of muscle contraction. They respond to the stimuli originating from the muscle they belong to, so called proprioceptors, which provide information about position of body parts in postures and movements to the central nervous system.

Basically, each spindle consists of a few, small, specialized intrafusal muscle fibres, which are innervated by both sensory and motor nerve end-ings. These fibres are surrounded by an expanded spindle capsule (Warwick and Williams 1971).

The nuclear bag and nuclear chain fibres are present in each spindle. The nuclear bag fibres are much longer, extending beyond the capsule to attach to the endomysium of the surrounding extrafusal muscle fibres. The poles of the nuclear chain fibres are attached to the capsule or to the sheaths of the nuclear bag fibres. Sensory innervation of the muscle spindles is of two types, the primary and secondary endings. The primary endings form a spiral enwrapping the nuclear bag and chain fibres around the central equator. The secondary endings are largely confined to nuclear chain fibres and are beaded peripherally on both sides of the primary endings. The ɣ-motorneurone endings in muscle spindles innervate the intrafusal fibres and by contracting them can cause sensibility adjustments of the spindle (Warwick and Williams 1971).

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In the human, only the jaw-closing muscles contain MSs. Jaw-opening muscles lack the MS. Also, unlike the spindles elsewhere in the body, spin-dles in the human jaw-closers contain very large numbers of intrafusal fibers per spindle (up to 36). This fact reinforces the idea, that jaw MSs should have a strong proprioceptive input to the jaw muscles and hence controlling the mastication (reviewed in Türker 2002). Furthermore, when spindle cell bodies were destroyed, the facilitation of the jaw-closers disappeared almost completely (Morimoto et al. 1999).

Cutaneous and mucosal receptors

A synaptic coupling between single orofacial afferents and the moto-neurones that innervate the masseter muscle confirms the importance of these afferents in the control of mastication (Türker et al. 2005). Ruffini endings, Merkel cells and free nerve endings are found in both cutaneous (hairy and glabour skin) and oral mucosa, while Meissner cells are absent in hairy skin (reviewed in Jacobs et al. 2002). Pacinian corpuscles are situated within the glabour and hairy skin and follicle endings are found only in hairy skin (reviewed in Jacobs et al. 2002).

Temporomandibular joint receptors

The receptor types found in the temporomandibular joint capsule include free nerve endings, Ruffini endings, Golgi organs, and Vater-Pacini cor-puscles. It has been claimed that the Ruffini endings and the Golgi organ within the capsule function as static mechanoreceptors, the Vater-Pacini endings as dynamic mechanoreceptors, and the free nerve endings as the pain receptors (reviewed in Türker 2002). The results of the research which has been done on the connection of these afferents to masticatory moto-neurones are very obscure and the research was done only in animals.

Tendon organs

There is only limited evidence for the existence of tendon organs in human or animal jaw muscles (Matthews 1975). The functional connections of these afferents to the masticatory muscles are not known.

1.2. Features of human masticatory system experiments

Previous studies to indicate the importance of intra- and perioral recep-tors in feedback control of mastication can be criticized since they have

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been performed either on anaesthetized animals or on human subjects using only the probability-based analyses.

1.2.1. General anaesthesia

Many experiments on jaw reflexes have been conducted on anaesthetized animals (Bonte et al. 1993; Dessem et al. 1988; Hannam 1969; Lavigne et al. 1987; Morimoto et al. 1989). The use of general anaesthetics is likely to give erroneous results as general anaesthetics work by means of directly affecting the cell membranes and synaptic transmission between neurones (Nicoll 1972; Nicolson et al. 1976). Such preparations also lack supraspinal inputs that normally help activate the interneurones that are essential for the completion of reflex circuitries (Matthews 1972). Therefore, functional information on neuronal pathways obtained from reduced animal experi-ments may be misleading.

1.2.2. Excitatory common drive

Consciousness human subjects can control some of the experimental conditions or respond to them and help the researcher to discover neuro-physiological mechanisms, which cannot be investigated in animals (for ex., it is well known that an excitatory common drive changes reflex responses dramatically and the control of excitatory drive is possible only in human experiments). The constant level of activation of jaw muscles is important in the reflex studies.

The level of muscle background activity or excitatory drive (i.e., in the prestimulus interval) in humans must be controlled in order to make quanti-tative reflex measurements (Türker and Miles 1989). Jaw reflex response changes according to the level of the common drive. The reduced inhibition of muscle activity has been observed at higher bite forces (Türker 1988) or at higher firing rates of the control SMU (Türker and Miles 1989). A smaller SMU inhibitory reflex was observed when the SMU fired at higher rate (Miles and Türker 1986; Miles et al. 1987). Reflex work where slowly rising mechanical stimuli were applied to the incisor teeth in an orthogonal direction suggested that, when subjects maintained high activity of a masseter muscle, the output closing force was a net reduction whereas at low levels of background activity, the stimulus generates a net closing force (Yang and Türker 1999). The peak-to-peak amplitude of a jaw-stretch reflex was significantly dependant on the clenching levels with the larger amplitudes of the reflex during higher clenching level (Wang and Svensson 2001). The higher level of muscle activity maintained by the subject

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kedly reduced both the incidence and the duration of the inhibition produced by innocuous and noxious stimuli of intraoral and facial regions (Yu et al. 1973). The control of common drive can be achieved by using on-line SMU firing rate as an acoustic or visual feedback.

The advantage of using human subjects in our experiments was that they could keep the common excitatory drive of the SMU constant, i.e. the firing rate of the SMU could be kept at the same level during the experiment. Therefore, controlling the firing frequency of the SMU makes the recording of jaw reflex response reliable during the constant common drive. In animal experiments, common drive cannot be controlled, since most of these experiments were done with the anaesthetized animals (Morimoto et al. 1989).

1.2.3. Sleep changes the reflex response

During quiet sleep jaw reflex activity decreases: a clear decrease in jaw-opening reflex amplitude, a longer reflex latency and an increase in intra-cortical microstimulation thresholds for evoking rhythmic jaw movements and orofacial twitches suggest that the excitability of the jaw motor system is reduced during light non-rapid eye movement sleep in macaque monkeys (Yao et al. 2013). Nevertheless, the excitatory drive cannot be controlled during sleep and comparisons of the reflex parameters between awake and sleep conditions have to be done with care.

1.2.4. Indirect methods for the evaluation of the jaw reflex

Human experiments by using indirect methods help researchers to esti-mate neuronal circuitries and masticatory control mechanisms through the contribution of the PMRs, MSs and other trigeminal receptors. When stimu-lating primary afferents (the ones, which have their cell bodies within trigeminal ganglion (V Gang) or trigeminal mesencephalic nucleus (V Mes N) during normal experimental conditions, reflex can only be recorded from the terminal efferential component – the SMU. The relevant indirect methods which are the PSF and the PSTH techniques were combined together, since the PSF represents the duration, end and the later events of the reflex and the PSTH is better for evaluating the beginning of the reflex response (Piotrkiewicz and Kudina 2012; Türker and Powers 1999, 2003).

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1.2.5. Psychological factors

Psychological factors are important for human experiments. It is well known that attention, general fatigue, anxiety, emotions, motivation, all can influence the jaw reflex response. It has been demonstrated previously that the prediction of the stimulus onset may contribute to the occurrence of habituation in jaw reflexes (Desmedt and Godaux 1976) and it may influ-ence the incidinflu-ence and duration of inhibition (Yu et al. 1973). Nevertheless, a constant stimulus application was used in numerous reflex studies. There-fore, in our study mechanical stimuli were delivered to the teeth randomly between 0.8–1.2 s.

1.2.6. Local anaesthesia and the excitatory common drive

LA for the temporal blocking of the activity of the PMRs allowed us to investigate the contribution of the PMRs and MSs to the jaw reflexes and mastication (Brinkworth et al. 2003; Naser ud-Din et al. 2010; Türker and Jenkins 2000). While the periodontium is anaesthetized, a subject can still control the firing rate of the SMU via auditory feedback, therefore keeping the common drive constant. It is possible to compare the jaw reflex response before and during LA of the periodontium and the synaptic contributions of the PMR and MS to the masticatory muscles can be determined.

1.2.7. Reaction time

In human experiments, the latency of the conscious component of a res-ponse (reaction time) to a stimulus can be detected, which clearly deter-mines the after-stimulus interval for the uncontaminated reflex response. The minimum reaction time was found to be 140 ms for the masseter EMG and 150 ms for the bite force when 20 ms rising time axial stimulus to a tooth was used (Brinkworth and Türker 2005). In other study the minimum reaction time to tap stimulation and electrical lip shocks was found to be 80–90 ms and to push stimulation the minimum reaction time was 140 ms (Brodin et al. 1993a). Reaction time for temporalis muscle was found to be 103 ms, for the masseter muscle – 168 ms and for the bite force record – 165 ms (Brinkworth et al. 2004). Therefore, the conclusions about the reflexive changes in muscle activity after the reaction time must be done with care.

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1.2.8. Control of a jaw position during experiment

In human jaw reflex studies subjects were asked to bite onto rubber bung (Yemm, 1972a,b), cotton rolls (Yu et al. 1973) or to clench on their incisor teeth (McMillan, 1994) to obtain continuous jaw muscle activity. However, different reflex responses were registered during various dental occlusions (Wang and Svensson, 2001). The conclusion that there were no differences in reflex behaviour in different muscle regions may be misleading when the SMU reflex behaviour in different masseter locations was investigated during only an anterior occlusion (McMillan, 1994). During different occlu-sions different oral and perioral receptors may be activated, therefore the jaw position must be controlled carefully during jaw reflex experiments to obtain reliable results. In our studies for the stabilization of the jaw position during experiments we have used stainless-steel bite bars and individual dental impressions for both upper and lower jaws. Dental impressions on the bite bars were made when a subject maintained one’s centric relation posi-tion of the jaws. This methodological consideraposi-tion allowed us to control afferential inputs from receptors such as PMRs, MSs and joint afferents during the experiments and to obtain reliable results.

1.2.9. Inter-subject differences

Occurrence, latency, strength, duration of the reflex was found to be significantly different between subjects and this illustrates that person-to-person variation is the source of the majority of differences observed (Brinkworth et al. 2003; Brinkworth et al. 2004; Brinkworth and Türker 2005). Tooth separation, tooth angle, periodontal health, recent tooth usage and the amount of ɣ-motoneurone activity present may be important and these factors may have the effects on the reflexes of the jaw muscles (Brinkworth et al. 2003; Brinkworth and Türker 2005).

1.3. Stimuli used in human jaw reflex studies

1.3.1. Stimulation of a face region

In humans, masticatory system has been studied by different means of afferent stimulation and recording of the response to that stimulation from the jaw muscles (reviewed in Türker 2002; Türker and Miles 1989). The classical stimuli used for this purpose are electrical and mechanical stimuli. Also, researches have used acoustic stimulation and recorded jaw muscle

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reflex responses (Deriu et al. 2005). Mechanical stimuli can be applied to the teeth, oral mucosa or to the jaw. To investigate the reflex control of the jaw muscles, afferent stimulation of the lips, palate, gingiva or sensory nerve is used by the means of electrical stimulation. The type of stimulus is selected according to the receptor or afferent localization, specialization, and the aim of the investigation. Because of rich facial and oral inner-vations, the selective stimulation of a certain group of the receptors is challenging in the human research. Therefore, the type of the stimulation, the parameters of a stimulus and the point of application of the stimulus are chosen deliberately.

1.3.2. Advantages and disadvantages of various stimuli

Mechanical and electrical stimuli have their advantages and disad-vantages which must be evaluated for a good experimental design and a correct interpretation of the results since the methods of the investigation of the human masticatory system are indirect (reviewed in Türker 2002). The main disadvantages of the mechanical teeth stimulus are these: it is difficult to control and keep the same stimulus parameters (strength, duration and profile of the stimulus); it is difficult to stimulate the same site within every stimulus; and if the stimulus is large enough to initiate jaw reflex, it can stimulate other receptor systems others than for ex., PMR system. A tap to the skin of the face stimulates the cutaneous receptors in the area of application, the vibration-, stretch-, and position-sensitive receptors in and around the jaws, and also the vibration-sensitive receptors in the inner ear (reviewed in Türker 2002).

Electrical stimuli have the following advantages over mechanical stimulation: easy to control stimulus parameters (intensity, duration, fre-quency, profile); since it do not induce vibration, stimulus affects only the close stimulation site; there are no movement artifacts and hence position-sensitive receptors within the jaw muscles and temporomandibular joint are not activated (reviewed in Türker 2002; Türker and Miles 1989).

1.3.3. The properties of mechanical teeth stimulation: preload

The reflex connections of the PMRs are normally studied using mecha-nical stimulation to the tooth in orthogonal or axial direction. However the profile of the stimuli has been subject of criticism as the exact stimulus profile that was delivered to the tooth was not precisely controlled in most experiments (for review: Türker 2002). Therefore, resulting reflex response could not be compared with the experiments where stimulus profile was

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recorded and regulated using a Proportional-Integral-Derivative controller system to deliver exact stimuli each time. If the physical relationship between the stimulating probe and the stimulated tooth changes, the rate of rise of the stimulus and stimulus profile will also change (Türker et al. 2007b). It is therefore the best to physically connect the probe to the tooth to be stimulated by applying preload to eliminate slack between the opposing surfaces (Türker et al. 1997b). Preload on a tooth stimulated not only elimi-nates high frequency components caused by a stimulus, but also can change reflex response. The lowest preloads caused the largest inhibitory perio-dontal-masseteric reflex responses, followed by ‘excitation’ (Sowman and Türker 2008). The PMRs can reflexively alter bite force during biting tasks only where the force exerted is small and this was evidenced by the sharp decline in evocable reflex activity when the tooth was pre-loaded above 1 N (Sowman and Türker 2008). The occurrence rate of inhibition decreased when the stimulated tooth preload has been changed from 0.5 N to 1.5 N during 10% maximum voluntary contraction and the increasing of the sti-mulus force from 1 N to 3 N increased the occurrence rate of inhibition from 23% to 64%, respectively (Brinkworth and Türker 2005). It was therefore proved that an exact stimulus profile could only be applied to a tooth if a preload was used (Türker et al. 1997b).

1.3.4. Stimulating different groups of PMRs

Fast-rising and slow-rising mechanical teeth stimuli are used to study PMR synaptic connections with the motoneurones of jaw muscles. Each of these stimuli preferentially activate different groups of the PMR receptors: the fast-rising (‘tap’) stimulus (i.e. stimulus with a 1000 N/s or greater rate of rise) stimulates rapidly adapting PMRs, while slow-rising (‘push’) stimuli (i.e. stimuli with a 40 N/s or lower rate of rise) activates slowly adapting PMRs (Trulsson and Johansson 1994; Hannam 1969, 1976). The slower pushes may preferentially activate the slowly adapting receptors, which could send a sustained, lower-frequency output to the masseter moto-neurones (Hannam 1969) along a separate, excitatory interneuronal path-way. The slowly adapting receptors in particular are capable of signaling small changes in the bite force (Hannam 1976).

Notwithstanding, it is also declared that human periodontal mechano-receptive afferents innervating the anterior teeth are all slowly adapting (Trulsson and Johansson 1996) and only the localization of the PMRs within the periodontium determines the reflex output to the motoneurone (for more details, see 1.1.2.). However, ramp forces and taps applied to the teeth and gingivae cause different reflex responses in jaw closing muscles, although

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all of these responses were only inhibitory (Bonte 1993). Nevertheless, fast force rate sensitive PMRs induces a powerful inhibitory jaw reflex, while slow force rate applied to the teeth generates excitatory responses for the masseter muscle (Amano and Yoneda 1980; Brinkworth et al. 2004; Brodin et al. 1993b; Funakoshi 1981; Lavigne et al. 1987; Lund and Lamarre 1973; Tucker and Türker 2001; Türker et al. 1994, 1997b; Yang and Türker 1999).

1.3.5. Stimulating different groups of teeth

The mechanical stimulus strength to elicit jaw reflex varies between the groups of the teeth: the force of at least 6 N applied to the molar teeth is required to elicite reliable jaw-muscle reflexes and considerably lower forces to the incisors (about 0.25 N) elicit reflexes in jaw-closing muscles (Brinkworth et al. 2004). The increasing sharp mechanical stimulus strength increases the strength of periodontal inhibitory and spindle excitatory response (Naser Ud-Din et al. 2010). However, inhibitory effect overcomes the excitatory effect arriving from MSs and the excitatory response can be only seen during local anesthetic block of periodontium.

1.3.6. Fast clench

From a functional point of view, the location of the receptor does not create a problem, as long as they respond to tooth movement (Hannam 1976). A fast clench is a natural way of stimulation of the PMRs and can be registered by using occlusal scanning system. Although, possibly, many different groups of the trigeminal receptors are activated during a sudden clench, the net response from all the jaw-closers may show the power of the receptor input to the jaw-closing muscles. This information can be clinically useful while assessing the results of the rehabilitation of the masticatory system or for the diagnosis of the dysfunction of the masticatory system.

Therefore, stimulus profile must be precisely controlled during the experiments for obtaining reliable results. By precisely controlling the stimuli, it is possible to stimulate different receptors within the masticatory system and to determine their contribution to the jaw reflex. Presence of the preload and exact stimulus force profile affects the success of the stimulus to elicit a certain type of jaw reflex response (Türker et al. 1997b).

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1.4. Recording techniques as tools to study human jaw reflexes

1.4.1. Surface electromyography technique

Reflex responses of the human masticatory muscles to mechanical or electrical intra- or perioral stimuli classically are recorded using electro-myogram (EMG) technique. The most common and widely used EMG technique for human jaw reflex studies is the SEMG (van der Bilt et al. 1997; Brinkworth et al. 2003; Brodin and Türker 1994; Brodin et al. 1993b; Cadden and Newton 1988; Godaux and Desmedt 1975; Haßfeld et al. 1995; Hück et al. 2005; Sowman and Türker 2008; Sowman et at. 2010; Tucker and Türker 2001; Türker and Jenkins 2000; Yang and Türker 1999; Yemm 1972a, b; Yu et al. 1973; Wang and Svensson 2001).

The changes evoked in the SEMG by a stimulus are used to determine the relationship between the stimulated afferent system and the motor-neurones of the muscle under study (Türker and Miles 1989). In many studies, the probable reflex effect of the stimulus has been evaluated by observing single traces or superimposed traces of the SEMG (Haßfeld et al. 1995, Yu et al. 1973). However, it is clear that measurements from individual traces are subject to significant errors, since the latency and the duration of the response cannot be measured accurately (Lavigne et al. 1983). The reliability of SEMG records is substantially improved if the repeated trials are full-wave rectified and averaged (Miles and Türker 1987; Türker and Miles 1989; Yemm 1972b).

Recording of the SEMG does not always indicate the activity of the target jaw muscle. Other muscles in the proximity of the muscle investigated by the SEMG technique do contribute to the general electrical activity (Türker and Miles 1990). For example, most reflex studies on jaw reflexes utilized the masseter muscle SEMG. This muscle is however cove-red by several layers of mimic muscles such as the zygomaticus major, platysma and risorius (Warwick and Williams 1973) which are activated without conscious intention of the subject. These mimic muscles are activated especially when the subject is anxious about the experimental procedure, fatigued, in pain, uncomfortable or simply because he/she un-intentionally moves the face or blinks. The subtle psychomotor effects reflect on the SEMG of the face region and imagining happy or sad events causes measurable differences in the corrugator SEMG (Oliveau and Willmuth 1979). Therefore, the EMG activity recorded using surface electrodes over the masseter can come from any of these mimic muscles as well as from the masseter itself. It is hence suggested that researchers consider these limitations when evaluating the SEMG from the masseter.

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1.4.2. Single motor unit technique

The central nervous system does not operate ‘in muscles’, i.e. a muscle is never activated as a whole. Instead, the central nervous system activates individual motor-units. Thus, the motor unit is the basic unit of motor activity. It consists of a single α-motor neuron and the set of muscle fibres innervated by this neuron. A given muscle fibre belongs to only one motor unit. When a motor unit is activated, all of its fibres are activated and produce a force.

Recording of the electrical activity from the single muscle fibres using intramuscular electrodes (Fig. 1.4.1), called SMU recording, was introduced to human neurophysiological studies together with the specific analysis techniques as a precise and reliable method for investigation of the motoneurone PSP.

Fig. 1.4.1. Intramuscular silver fine-wire electrodes for the recording

of the SMU (single motor unit) activity.

The SMU reflex responses of the masseter and the temporalis muscles while stimulating orofacial afferents have been documented in many studies (Brinkworth and Türker 2005; Bonte 1993; McMillan 1994; Miles et al. 1987; Türker et al. 1994, 2006; Yang and Türker 2001). Unlike the SEMG technique which in effect assumes that all the SMUs belonging to the muscle under study have similar reflex responses at a given time, the reflex responses of individual motor neurones can be investigated without preconceived assumptions (Türker and Miles 1989).

SMU recording of the jaw muscles is minimally invasive method, because a needle used to introduce the electrodes within the muscle is

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removed immediately after the insertion of the electrodes leaving the ‘fish-hook’ fine-wire silver electrodes within the muscle. After an experiment, the electrodes are gently pulled out with ease.

The SMU technique includes many advantages when compared with the SEMG technique: an increased precision in measuring the timing of neural events (since SMU potentials are effectively binary or ‘ON/OFF’ events); increased precision in the control of pre-stimulus excitation of the motor-neuronal pool; the ability to quantitate the reflex changes evoked; and the ability to determine the amplitude and time-course of the excitatory postsynaptic potentials (EPSPs) and the IPSPs in human motor neurones (Türker and Miles 1989). The SMU recording enables the evaluation of reflex responses in the synaptic level accordingly neuronal pathways of the reflex can be determined. The SMU technique avoids many pitfalls of the SEMG technique, and enables unambiguous conclusions to be drawn concerning the nature of synaptic relationships between afferents and motor neurones (Türker and Miles 1989).

1.5. Analysis methods of the jaw reflex responses

1.5.1. Probability-based analyses

Although synaptic potentials cannot be recorded directly from human motoneurones, their characteristics can be inferred from measurements of the effects of activating a set of peripheral or descending fibres on the discharge probability of one or more motoneurones. These effects often are assessed by compiling a PSTH, which measures the probability of occur-rence of a motoneurone spike as a function of time from the stimulus (Calancie and Bawa 1985; Garnett and Stephens 1980).

To the best of our knowledge, all previous works on human jaw reflexes have utilized probability-based analyses such as the stimulus triggered averaging of the SEMG, the PSTH and raster dots of SMU spikes (reviewed in Türker 2002). Recent experiments on rat brain slices have shown that the probability-based analyses are subject to significant errors (Türker and Powers 2005). The errors of probability-based techniques are synchro-nization-related errors, count-related errors and number-related errors. All these errors belong to the SEMG technique and the first two belong to the PSTH technique. These classical analyses techniques rely upon the number of occurrence of action potentials at a particular time after the stimulus. It therefore generates significant errors as the action potentials are phase advanced or delayed by the stimulus-induced synaptic potential (‘count-related errors’). On the other hand, frequency-based methods are free from

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such errors as they only consider the changes in the instantaneous discharge rate of the spikes that are not affected by the density of spikes at any particular time after the stimulus (Türker and Cheng 1994; Türker and Powers 2003).

An increase in the poststimulus SEMG, preceding an inhibitory phase, may be an artifact of the averaging process (Widmer and Lund 1989). An increase after a decrease in SEMG records can be simply a cluster of delayed action potentials by the preceding inhibition (Miles et al. 1987) (‘count-related error’). Any such clusters of action potentials related to the stimulus will fire again at about one interspike interval and hence induce several peaks and troughs. These changes in the SEMG may be described wrongly as an excitatory or an inhibitory connection of the stimulated afferent to the motoneurones (Awiszus et al. 1991; Bonte 1993; Hück et al. 2005; Türker and Cheng 1994). This is termed as the ‘synchronization-related error’.

The SEMG also has one other major pitfall, ‘number-related error’, where a large EPSP shadows a later EPSP because many of the active motoneurones discharge in response to the earlier EPSP can no longer fire for a later one. This period resembles a ‘silent period’ or a period with reduced activity on the averaged graph, and the EPSP underlying the period may only be examined by the discharge rate of the SMUs in the muscle (reviewed in Türker 2002).

For the above reasons, it is therefore possible to consider only the very first response in the SEMG as the true representative of the PSP in the motoneurone. The peaks and troughs that follow the very first response in the averaged SEMG records should therefore be interpreted with extreme caution.

However, the PSTH is subject to the same ‘synchronization-’ and ‘count-related errors’ as the SEMG, since both of these analyses depend upon the probability of spike occurrences. Therefore, again, other than the very first response in the averaged record, one cannot take the late responses to represent the stimulus-evoked synaptic potential in the motoneurone pool.

1.5.2. Frequency-based analysis

The PSF plots the instantaneous discharge frequency values against the time of the stimulus and recently has been used to examine reflex effects on motoneurones (Türker and Cheng 1994), as well as the sign of the net common input that underlies the synchronous discharge of human motor units (Türker et al. 1996).

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The discharge frequency of a motoneurone reflects the net current reaching the soma (Baldissera et al. 1982; Powers et al. 1992; Redman 1976; Schwindt and Calvin 1973), thereforeany significant change in the poststimulus discharge frequency should indicate the sign and the profile of the net input (Türker and Cheng 1994; Türker et al. 2005).

The PSF avoids many of the errors associated with the interpretation of the PSTH features. The PSTH does not indicate the duration of the inhibitory event because when the delayed spikes finally occur, they induce a large increase in the discharge probability in the PSTH that has been wrongly interpreted as an excitatory event. This point has been discussed widely in the human reflex literature (Miles and Türker 1987; Miles et al. 1987). Unlike the PSTH, the PSF does indicate the rising phase of the IPSP and hence indicate the total duration of the IPSP for slower IPSPs.

Based on the PSF analysis, the initial phase of the IPSP appeared as a large gap in the record where very few or no discharges occurred. The subsequent phase of the IPSP was associated with frequency values that were lower than the background values. By either the EPSP or IPSP, the PSTHs contained secondary peaks and troughs that were not directly related to the underlying PSP, but instead reflected the regular recurrence of spikes following those affected by the PSPs (the source of ‘synchronization-’ and ‘number-related errors’). The PSF analysis is useful for indicating the total duration and the profile of the underlying PSPs, since the discharge frequency of the spikes which followed the PSPs very closely did not contain ‘synchronization-’ and ‘count-related errors’.

An EPSP produces an increase in the instantaneous discharge rate of the interval in which it occurs. However, this effect is not due to an analysis error but reflects a genuine change in the membrane potential of the cell as a result of an injected current (Türker and Powers 1999).

The PMRs as well as the MSs and most likely other trigeminal afferents play an important role on the control of human mastication. But the extent of this role has not been clarified yet since the SMU technique and the PSF combined with the PSTH analysis techniques were introduced recently for the investigation of human masticatory system.

1.6. Peripheral control of mastication. Reflexes elicited in jaw muscles by mechanical stimulation of the teeth

Mechanical teeth stimulation enables us to investigate jaw reflexes of the origin of the PMRs and the MSs. Contribution of the PMRs and MSs to jaw muscles during the static and dynamic conditions has been widely studied (Ottenhoff et al. 1992a, b; Brodin et al. 1993; Türker et al. 1994; Svensson

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et al. 2000; Brinkworth et al. 2003; reviewed in Türker 2002; Sowman et al. 2010; Naser Ud-Din et al. 2010). However, most of the previous studies used SEMG signal of the masseter to investigate the feedback properties and neuronal connections of these afferents. As we have detailed above, SEMG results should be evaluated with caution as it includes not only the response of the jaw muscle (masseter) but also the responses of the mimic muscles to the stimulus tested.

Previous studies found different reflex responses under different experimental conditions. Mechanical teeth stimulation by means of tooth taps evoked inhibitory reflex responses in the jaw-closing muscles (van der Glas et al. 1985; Sessle and Schmitt 1972; Bjørnland et al. 1991; Brinkworth and Türker 2005). On the other hand, it is reliable to assume that PMRs contribute to the ‘positive feedback’ to the jaw-closing muscles (Amano and Yoneda 1980; Funakoshi 1981; Lavigne et al. 1987; Lund and Lamarre 1973) or tooth taps evoked inhibitory reflex responses in the jaw-closing muscles (van der Glas et al. 1985; Sessle and Schmitt 1972; Bjørnland et al. 1991; Brinkworth and Türker 2005). Slow- or rapid-raising stimuli applied to the teeth were used in different studies and various reflex responses have been elicited. The slowly rising stimuli mainly induce an excitatory reflex, whereas the rapidly rising stimuli usually induce inhibition (Brinkworth et al. 2004; Brodin et al. 1993b; Türker et al. 1994; Yang and Türker 1999).

Other variables, like the presence of a tooth preload and the exact stimulus force profile induce a certain reflex response (Brinkworth and Türker 2005; Türker et al. 1997b).

1.6.1. Early excitation

A tap stimulus usually elicits a brief early excitation followed by inhibition in masseter muscle (Goldberg 1971; Sessle and Schmitt 1972).

There is evidence that this excitatory response arrives from PMRs, however there is also evidence that this is not an excitatory response but the artifact induced by averaging a rectified EMG signal and reduced can-cellation of potentials adjacent to the subsequent inhibition (Widmer and Lund 1989).

An early excitatory response during axial tooth stimulation with the latency of 13 ms is most likely due to the activation of MSs in the jaw closers that respond to stretch and vibration. Nevertheless, this early excitation appeared only if fast rate stimuli were applied to a tooth, which have larger high frequency components which were not eliminated with tooth preload (Brinkworth at al. 2003).

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1.6.2. Late excitation

While using push stimuli for mechanical tooth stimulation, there was minimal short-latency inhibition response of masseter muscle, with the predominant response being an excitation beginning at about 60 ms latency and continuing for about 70 ms (Brinkworth et al. 2004; Brodin et al. 1993). Using SEMG technique and tap stimulus to the incisor, a peak in reflex response, which followed inhibitory trough was interpreted as a true tatory response with a latency of 54 ms (Brodin et al. 1993b). This exci-tation is a ‘count-related error’ and not a genuine reflex (Miles et al. 1987).

When a push stimulus was applied to the molar tooth, the main SEMG response was late excitation with the latency of about 57 ms and increased bite force (latency 60–70 ms); the response was not abolished by LA (Brinkworth et al. 2004).

1.6.3. Inhibition

Characteristics of the SEMG reflex responses

The main response for a tap to the teeth was inhibitory with a latency of about 10–20 ms, which depended on the used recording technique (Brinkworth and Türker 2005; Brodin et al. 1993b; Bonte et al. 1993; Dessem et al. 1988; Sato et al. 1994; Sessle and Schmitt 1972; Türker and Jenkins 2000; van der Glas et al. 1985, 1988; Yang and Türker 1999).

When using the SEMG technique, the average latency of inhibition to axial stimulation of an incisor was found to be 21 ms and bite level and stimulus force influenced changes in the latency for 1.6 ms (Brinkworth and Türker 2005). Latency of inhibition of about 21 ms coheres with others’ findings for both horizontal loading (Yang and Türker 1999) and unloading (Türker and Jenkins 2000), and with work on axial stimulation (Brinkworth et al. 2003) when SEMG technique was used. The discrepancy between the durations of the reflex when axial stimulation was performed to a tooth (21 ms) and when orthogonal direction to stimulate a tooth was used (37 ms) can be explained by the importance of rate and/or direction of force application (Brinkworth et al. 2003).

Similarly innocuous mechanical stimulation of the lip produced a period of complete or partial inhibition in the masseter muscle activity with a latency of 15–20 ms and a duration of 8–18 ms (Yu et al. 1973). Although in the same study of Yu et al. (1973) noxious lip stimulation produced a longer latency (40–50 ms) inhibitory response and dependently on the level of background muscle activity a duration varied between 15 ms and 55 ms.

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Although when a tap is applied to a tooth, one inhibitory phase was detected in majority studies, some researchers detected two distinct phases of inhibition with latencies of 10 ms and 45 ms (van der Glas et al. 1985).

Characteristics of the SMU reflex responses

The latency of inhibition increased and the strength of inhibition decreased when stimulus rise time was slower, although the reflex duration did not change (Brinkworth et al. 2003). The duration of inhibitory period of temporalis muscle SMUs in cats depended on both the amplitude of the stimulation force and the rate at which the force was applied (Bonte et al. 1993). The duration of the inhibition was significantly longer in the cumulative peri-stimulus dischargegram record (27 ms) than in either the intramuscular EMG (21 ms) or the SEMG (20 ms) (Brinkworth et al. 2003; Brinkworth and Türker 2005).

The latency of SMU inhibition in masseter muscle was 10 ms when the PSTH analysis was performed and it was declared that the prestimulus firing frequency of the SMU determines the duration of the inhibition (Bonte 1993). Slowly firing SMU had the duration of inhibitory period of 60 ms and fast firing SMU had the duration of inhibition of 20 ms in the PSTH (Bonte 1993). Also the same study demonstrated that for slow firing SMU, a second inhibitory phase was inherent, which lasted for about 30 ms. The apparent excitation seen in the histograms was most likely due to syn-chronized reactivation of the silenced SMU spikes (Bonte 1993).

The strength of the inhibition was larger in the intramuscular EMG and the cumulative peri-stimulus dischargegram recordings than in the SEMG (Brinkworth and Türker 2005).

1.6.4. The unloading reflex

The unloading mechanism in the jaws is experimentally studied by slowly introducing, then suddenly and unpredictably removing, a load bet-ween the jaws (revieved in Türker 2002; Türker et al. 2007b). The most prominent component of the jaw-unloading reflex is a short-latency (10–20 ms) reduction in the SEMG of the jaw closing muscles (Lamarre and Lund 1975; Türker and Jenkins 2000). In addition, an increase in the SEMG of the anterior digastric muscle is observed at a slightly longer latency (appro-ximately 25 ms) (Lamarre and Lund 1975), although in one study there was no measurable pattern detected in the poststimulus changes in the SEMG of the anterior digastric muscle in response to tooth unloading (Türker and Jenkins 2000). In some studies blocking periodontal input by infiltration of

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LA around the roots of the teeth, failed to alter the unloading reflex (Lamarre and Lund 1975; Poliakov and Miles 1994; Miles at al. 1995). With a technique, which allows to keep the jaw appreciably stable, it was demonstrated that unloading inhibitory reflex was totally abolished by the LA block of the stimulated teeth, hence indicating that PMRs do contribute to the jaw-unloading reflex (Türker and Jenkins 2000).

When using unloading stimulus on a tooth, three identifiable reflex components were observed in the masseteric SEMG-CUSUM: the first component was a weak short-latency excitation; the second was a prominent inhibition and the final reflex response was a long-latency excitation (Türker and Jenkins 2000). The later was not analyzed in the study since in represents the tail end of the IPSP rather than an EPSP (Türker and Powers 1999). The latency of excitation was 13 ms in both before periodontal LA and during LA conditions and it suggests the MS origin of the response. The incidence and the size of this early excitation increased when an incisor was unloaded during LA. The latency of an inhibitory response was 19.5 ms before LA and it abolished during LA, hence indicating that it is of periodontal origin.

Clinically, fracturing of an object between the jaws would unload at least one tooth in the upper jaw and one in the lower jaw in axial directions and also this axial unloading through interproximal contacts would orthogonally unload adjacent teeth and stimulate many more receptors than during a single tooth unloading experiments, therefore the unloading reflex effect on jaw muscles would be much more powerful.

1.6.5. Jaw-stretch reflex

In all previous studies on the human jaw muscle stretch reflexes, the spindles were activated using a sudden downward perturbation of the mandible (Poliakov and Miles 1994; Miles et al. 1995). The connection of jaw MS to the motoneurones that innervate jaw muscles were found to be monosynaptic excitatory. In these studies it was found that unlike the limb muscles, jaw muscles do not illustrate long latency excitation (Poliakov and Miles 1994) and the jaw-stretch reflexes are observed in only 35% of the masseter motor units tested (Miles et al. 1995), and stronger jaw-stretch reflexes are obtained during clenching with unilateral tooth support (Lobbezoo et al. 1993). Biting position also influence jaw-stretch reflex as the higher amplitude reflexes were recorded ipsilaterally of working side or with the increase of vertical dimension (Wang and Svensson 2001). The LA of the teeth of subjects biting on the levers of a loading apparatus increased the size of the jaw-stretch reflex (Lamarre and Lund 1975). When the

THE REFLEX RESPONSES IN HUMAN MASTICATORY SYSTEM: MASSETER MUSCLE SINGLE MOTOR UNIT AND BITE FORCE REFLEXES

Paulius Uginčius